Development of molecular methods for the identification of the main Cystic Fibrosis pulmonary pathogens

Respiratory infections remain a major threat to Cystic Fibrosis (CF) patients. In spite of the plethora of classical and molecular techniques available for the detection and identification of bacteria responsible for these infections, most of these methods lack either sensitivity or specificity. Thus, the aim of this work was to set up new molecular methods for the identification of the main CF pulmonary pathogens at different taxonomic levels: I) The first one was a method based on SNuPE (Single Nucleotide Primer Extension). A set of primers of different length specific for four pathogens (Pseudomonas aeruginosa, Staphylococcus aureus, Stenotrophomonas maltophilia and Burkholderia cepacia complex (BCC) bacteria) were designed on 16S rRNA gene and used either in simplex or multiplex SNuPE reactions using the DNA of the four pathogens. The SNuPE profiles obtained allowed the easy identification of isolates belonging to the four species. II) The second one was a method to identify Ralstonia and Burkholderia strains by applying a new molecular strategy based on the use of nested overlapping genes. The analysis of 1098 prokaryotic genomes revealed the insertion of marC, a gene unrelated to histidine (his) biosynthesis in the his operon, whereby marC partially overlaps with hisH only in all strains belonging to these two genera. Hence, three forward PCR primers (one for Burkholderia, one for Ralstonia and one for both genera) targeting marC and one reverse primer targeting hisH were designed and tested on Burkholderia and Ralstonia strains and the expected amplicons were obtained for all strains analyzed. All other species tested remained negative; III) The last method was based on SNuPE and might allow the identification at the species level of strains belonging to the 17 different species of the BCC. To this purpose, a set of primers were design on genes of the histidine (his) operon and their use in simplex or multiplex SNuPE reactions is in progress. The development of the three systems directly on CF patients sputum is in progress